Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

NPFF may participate in the regulation of blood pressure because it is present in the cardiovascular regulatory center in the hypothalamus 15 .The intracerebroventricular 16 , intranuclear tractus solitarius 17 , or intrathecal 18 administration of NPFF increases blood pressure, indicating that NPFF in the central nervous system increases blood pressure.The intravenous administration of NPFF also increases blood pressure 19 although it cannot cross the blood-brain barrier 20 .Therefore, both central and peripheral mechanisms contribute to the NPFF-mediated increase in blood pressure [15][16][17][18][19][20][21][22] .
The NPFF network in the hypothalamus is impaired in hypertensive patients 23 suggesting that the interaction of the NPFF system with other neurotransmitter system (s) could play an important role in the regulation of blood pressure.The dopaminergic system in the kidney is known to regulate blood pressure [4][5][6][7][8]24,25 , however, it is not known whether NPFF can regulate blood pressure in the kidney, and whether or not there is a functional interaction between NPFF and the dopaminergic system in the kidney.
The main objective of this study is to determine the presence of the NPFF system in the kidney, the physical and functional interaction between the NPFF system in renal proximal tubule cells (RPTCs) by determining their cAMP production, a known signaling pathway mediated by dopamine receptors, and the renal-mediated blood pressure regulation by NPFF and its receptors, NPFF-R1 and NPFF-R2.
The presence of NPFF in the mouse serum and kidney was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 2); the NPFF concentrations were 0.936 ± 0.22 pmol/g of tissue (n = 3) in the kidney and 541 ± 32 pmol/L (n = 3) in the serum of C57BL/6 mice.Next, we determined whether NPFF produced in mRPTCs is functional.As shown in Fig. 3, NPFF inhibited the forskolin-induced increase in cAMP production in mRPTCs in a concentration-(Fig.3a) and time-(Fig.3b) dependent manner.By contrast, RFRP-2, an RF-amide-related peptide with low affinity to NPFF receptors 28 , had no effect on forskolin-induced cAMP production (Fig. 3c,d).The ability of NPFF, but not RFRP-2, to decrease the forskolin-induced increase in cAMP level measured in cell culture supernatants (Fig. 3) was also observed in mRPTC lysates (Supplementary Fig. S3).

Antagonism between NPFF and dopaminergic systems in the kidney
We next investigated the potential physiological/pathophysiological effect of the interaction between the NPFF and the D1-like dopaminergic systems in the kidney.As expected, the D1-like receptor agonist, FEN [4][5][6] , increased intracellular cAMP production in hRPTCs; NPFF (10 −11 M) impaired the stimulatory effect of FEN at a concentration which, by itself, did not affect cAMP concentration (Fig. 6a).Similarly, the NPFF-R1 agonist, RFRP-3 (Fig. 6b) and NPFF-R2 agonist, AC-263093 (Fig. 6c), by themselves, did not induce changes in the cAMP concentration but they attenuated the stimulatory effect of FEN on intracellular cAMP concentration, indicating that both NPFF-R1 and NPFF-R2 can antagonize the stimulatory effect of FEN on cAMP production.

Discussion
NPFF, like dopamine, was originally found in the brain, but subsequent studies showed its synthesis in peripheral organs [9][10][11][12][26][27][28] . The curent study demonstrated that NPFF and its receptors are expressed in the brain and the kidney.NPFF is an endogenous neuropeptide that is predominantly present in the mammalian CNS and is implicated in pain modulation by regulating opioid signaling; other physiological functions are also affected, including the regulation of blood pressure 15 .The distribution and expression of NPFF and its receptors in the CNS have been studied in rats, mice, and humans [10][11][12][26][27][28][36][37][38][39] .Our data also revealed extensive distribution of NPFF-R1 and NPFF-R2 in the brain, particularly in the hypothalamus, including OVLT, SON, ARC, PVN, and HP.NPFF-R1 had relatively greater expression in ARC than PVN, while NPFF-R2 had similar strong expression in both ARC and PVN and other hypothalamic areas.The distribution of NPFF receptors in the brain is consistent with previous reports in mice 10 .In rats, strong Npff-r1 expression is observed in the lateral septum, PVN, VMH, DMH, HP, thalamus, amygdala, olfactory bulb, and medulla oblongata; low to moderate expression is observed in the dorsal motor nucleus of the vagus, substantia nigra, and locus coeruleus 11,28,36,39 .Npff-r2 is highly expressed in the hypothalamus, medulla oblongata, piriform cortex, lateral parabrachial nucleus, thalamus, lateral lemniscus, and trigeminal nucleus 11,28,36,39 .The expression of Npff receptors in dopaminergic, NPY, and other neuroendocrine-related neurons in PVN and ARC of hypothalamus areas 15,[38][39][40] may explain the involvement of Npff receptors in central blood pressure regulation, which warrants further investigation.
The expression and distribution of NPFF-R1 and NPFF-R2 in peripheral tissues, especially in the kidney, are not fully known.Limited mRNA expression of Npff is observed in the pancreas, lung, spleen, heart, adrenal gland, and skin 11,12,26,39 .The pancreas and adipose tissues have a considerable Npff expression in mice 41 , consistent with its role in the regulation of glucose and lipid metabolism 41,42 .Detectable levels of Npff -r1 mRNA expression are also evident in the adrenal gland, eye, intestine, kidney, lung, ovary, and spleen 11,12,28 .By contrast, Npff -r2 mRNA is highly expressed in adipose tissue, heart, kidney, retina, salivary gland, stomach, and urinary bladder 11,12,39 .Recent single-cell RNA sequencing showed considerable NPFF-R1 and detectable NPFF-R2 expression in hRPTCs (www.prote inatl as.org).However, considerable discrepancies in NPFF receptor expression exist in studies in CNS and peripheral tissues that may be due to the use of different methods and species 9,11,12,26,28,37,39 .NPFF receptors are speculated to be expressed highly on the plasma membrane because they are G proteincoupled receptors.However, we found that NPFF-R1 and NPFF-R2 were distributed in both plasma membrane and cytoplasm in human and mouse RPTCs.The predominant cytoplasmic distribution of NPFF-R1 and NPFF-R2 is consistent with that observed in gonadotropin-releasing hormone neurons 43 and epididymal white adipose cells 42 .Of note, the few NPFF receptors that overlapped with DAPI in our immunofluorescence study could indicate their potential nuclear localization, which needs further investigation.
The transcripts and proteins of NPFF and its receptors are expressed in human and mouse RPTCs, enabling NPFF to function as an autocrine system in the kidney, similar to the renal dopaminergic system [4][5][6] .The NPFF serum concentration in this study is similar to the NPFF plasma levels in mice and nonobese humans 41 but higher than that found in an earlier study in healthy humans 44 .The lower plasma NPFF concentrations could be caused by the use of frozen pooled plasma 44 , because NPFF is extremely sensitive to freeze-thaw cycles 41 .The circulating NPFF contains NPFF released from the spinal cord, the pancreas, and possibly the kidney or other unknown tissues 10,11,15,39,41,44 .Our study is the first report on kidney NPFF concentrations and renal NPFF signaling regulates blood pressure in mice.NPFF primarily exerts its functions via the Gαi/o protein which inhibits adenylate cyclase 13 , while NPFF-R2 can also couple to Gαs protein which leads to the stimulation of adenylate cyclase activity 14 .In mRPTCs, the observation that NPFF, RFRP-3, and AC-263093 attenuated forskolin-stimulated cAMP production corroborates the preferential linkage of these two NPFF receptors to Gαi/o.The time-and concentration-dependent inhibition of forskolin-induced cAMP production by NPFF further confirms a functional autocrine model of the NPFF system in RPTCs.In hypertension, sympathetic activity is increased whereas parasympathetic activity is decreased within the autonomic nervous centers in the hypothalamus and the brainstem 15 .Inhibition of adrenergic activity attenuates the increase in blood pressure caused by NPFF injection in the brainstem 16 , indicating a role of NPFF in the increase in sympathetic activity.NPFF levels are decreased in the DMV of human hypertensive subjects 23 and spontaneously hypertensive rats 45 , leading to a decrease in vagal activity and baroreflex dysfunction.The renal NPFF system could regulate blood pressure in the physiological state because physiological concentrations of NPFF (pM) decreased forskolin-stimulated cAMP production, and RF-9, the NPFF-R1 NPFF-R2 antagonist, reversed both RFRP3-(NPFF-R1 agonist) and AC263093-(NPFF-R2 agonist)-mediated decrease in cAMP production in RPTCs.Moreover, the renal subcapsular infusion of physiological concentrations of NPFF also decreased urinary sodium excretion and increased the blood pressure.Whereas scrambled peptide had no effect on the increase in blood pressure, RF-9 prevented the acute NPFF-mediated increase in blood pressure.The current observations are consistent with previous observations of others 18,29,46 .The intravenous administration of NPFF in anesthetized rats increased blood pressure that was attenuated by daY8Fa, an NPFF-R1 and NPFF-R2 antagonist 46 .In anesthetized rats, RF-9 almost completely abrogated the increase in blood pressure caused by the lateral cerebral ventricular injection of NPFF 29 or the intrathecal administration of NPFF 18 .These results indicate that the effects of NPFF on cAMP production and blood pressure were exerted through its own receptors.
However, the sodium transporters related to the involved NPFF receptors in the regulation of blood pressure in the kidney are not clear at this time.The protein expression of either NHE3 or Na + /K + -ATPase in Npff-r1 or Npff-r2 deficient kidney cortices in C57BL/6 mice fed with a normal salt diet cannot explain the NPFF-mediated increase in blood pressure.The RPT is the site where two-thirds of filtered sodium is reabsorbed; NHE3 is recognized to be the most important mediator of sodium transport across the luminal membrane of this nephron segment 47 .Dopamine has been shown to inhibit NHE3 activity by decreasing its expression and increasing its phosphorylation 48,49 .NPFF and the D1-like receptor agonist, fenoldopam, exerted a counter regulatory effect on cAMP production; NPFF-R1 and NPFF-R2, colocalized and physically interacted with D 1 R in the RPT.NPFF-R1, unlike NPFF-R2, also colocalized and interacted with the D 5 R in the RPT.
The renal dopaminergic system maintains normal blood pressure by increasing sodium excretion, especially in states of moderate sodium excess [4][5][6]50 . It s possible that the decrease in renal sodium excretion and increase in blood pressure due to the intrarenal administration of NPFF is related, in part, to its counter regulation of the renal dopaminergic system.In the kidney, the dopaminergic system also counter regulates the renin-angiotensin-aldosterone system [4][5][6]51 .We have also found that the angiotensin type I receptor co-immunoprecipitated with NPFF-R1 and NPFF-R2 in hRPTCs and infusion of NPFF and angiotensin II synergistically increased blood pressure in C57BL/6 mice (Asico LD and Jose PA, unpublished observation).In rats, the peripheral NPFFinduced increase in blood pressure was attenuated by blocking α1-adrenergic receptors 19 , indicating a potential interaction of the NPFF system with the adrenergic system.Therefore, the effect of NPFF on sodium excretion and blood pressure is probably related to the counter-regulation of a renal natriuretic system (dopamine) and/ or positive interaction with angiotensin II and the adrenergic system.NPFF weakly activates the MAS receptor in HEK293 cells 52 and promotes macrophage M2 polarization and prevents inflammation in adipose tissues 41 .Inflammation participates in the regulation of renal function and blood pressure 1,2,4-6,8 .Similar to Ang II, NPFF has a major effect on the increase in blood pressure, however, NPFF could also activate the MAS receptor and protect against an increase in blood pressure 52 .However, the role of NPFF in these interactions in the regulation of sodium excretion and blood pressure remains to be determined.
In addition to NPFF, a total of five groups of RF-amide peptides have been identified in mammals, including RF-amide related peptides (RFRP), prolactin-releasing peptides, kisspeptin, and pyroglutamylated RF-amide peptides 53 .Each peptide group has specific cognate receptors with considerable cross-reactivity with the receptors of the other groups 11,39,54 .NPFF has an affinity to both NPFF-R1 and NPFF-R2 11,17,39 .RF-9, a dual NPFF-R1 and NPFF-R2 antagonist, has been reported as a Kiss1 receptor agonist in the gonadotropin system 32 , while reduced circulating kisspeptin levels are observed in pre-eclampsia compared with normotensive pregnancy 55 .The acute intrarenal administration of RF-9, alone, had no effect on blood pressure but as aforementioned, its chronic renal subcapsular infusion slightly decreased the blood pressure.In RPTCs, both RFRP-3, an NPFF-R1 agonist, and AC-263093, an NPFF-R2 agonist, were able to inhibit the forskolin-stimulated cAMP production, suggesting that both NPFF-R1 and NPFF-R2 are involved in NPFF's inhibition of cAMP production.By contrast, RFRP-2, which, by itself, has a low affinity to NPFF receptors, did not affect the cAMP production, highlighting the specificity of NPFF action on NPFF-R1 and NPFF-R2.In the nucleus tractus solitarius, NPFF can bind to the RFRP receptor, which is co-expressed with neuropeptide Y (NPY) 54 .NPY and at least one of its receptors are present in RPTs 53 , whose activation can mediate the increase in blood pressure 56 .The intravenous administration of neuropeptide 26RFa, an agonist of GPR103, increases blood pressure, which can be attenuated by pretreatment with an antagonist of NPY receptor 57 .D1-like receptors can inhibit the vascular smooth muscle proliferation caused by NPY 58 .Therefore, it is important to determine whether NPY contributes to the NPFF-induced cAMP signaling and subsequent blood pressure regulation.
Despite efforts to obtain the most specific commercially available antibodies for the staining of NPFF receptors in both cells and kidney sections, the specificity of their staining still needs further confirmation.This is a particular concern with NPFF-R1 which has a weak staining in RPTCs and brain.Whether or not NPFF may function in the kidney through NPFF-R2 needs to be evaluated in a future study.It should be noted, however, that RNAscope and RT-PCR showed the expressions of Npff (NPFF), Npff-r1(NPFF-R1), and Npff-r2(NPFF-R2) mRNA in the RPT.
Recent genome-wide association studies demonstrated that a single nucleotide polymorphism (SNP) of NPFF, rs11170566, is associated with migraine, inflammation, and cardiovascular disorders 59 .SNPs of Npff-r1 may be related to glucose metabolism and growth-related traits of the common carp 60 .SNPs of Npff-r2 are associated with impaired lipid metabolism, obesity, and inflammation 61,62 .Moreover, epidemiological studies show an association of blood pressure with polymorphisms in the GPR10 receptor 63 , the cognate receptor for prolactin-releasing peptide, which may regulate blood pressure via NPFF-R2 64 .The association of SNPs of NPFF and its receptors with inflammation and aberrant metabolism of glucose and lipid is consistent with the notion that hypertension is a chronic inflammatory disorder 65 .Henceforth, studies are needed to determine if the gene variants of NPFF and its receptors can directly influence renal sodium transport and blood pressure.

Western blot
Western blotting was performed as previously described 69,71 .Briefly, kidney cortices were lysed in 1 × RIPA lysis buffer (Millipore, Billerica, MA), containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL), and the samples were adjusted to have the same protein concentration.The proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with primary antibodies and appropriately conjugated secondary antibodies.The images were visualized by a LiCor Odyssey Imaging system.

Immunofluorescence imaging
Immunofluorescence imaging was performed, as previously described 69,71,74 .HRPTCs or mRPTCs were grown on poly-d-lysine-coated coverslips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature.After washing with PBS, the cells, fixed on coverslips, were incubated with primary anti-NPFF, anti-NPFF-R1, anti-NPFF-R2, anti-D 1 R, or anti-D 5 R antibodies overnight at 4 °C.The coverslips were then incubated with the proper Alexa Fluor-488 and -555 secondary antibodies for 2 h at 4 °C.The coverslips were mounted in a proper antifade mounting medium and sealed onto glass slides.
Human kidney sections (Imgenex, San Diego, CA, USA) were prepared for antigen retrieval, using heat and pressure and immunostained for NPFF-R1, NPFF-R2, D 1 R, and D 5 R antibodies.Wheat germ agglutinin, conjugated with Alexa Fluor 647, was used to target the lectin-rich brush border and plasma membranes of RPTs.DAPI was used to visualize nuclei.For negative controls, the primary antibodies were replaced with normal rabbit serum at the appropriate dilutions.
Mouse brains were coronally sectioned at 10 μm thickness using a cryostat, and the sections, including the entire forebrain regions underwent standard immunohistochemistry, as previously reported 73 to study brain distribution of NPFF-R1 and NPFF-R2.For NPFF-R1 and NPFF-R2 immunostaining, the primary antibodies were diluted 1:100 in blocking buffer (3% BSA and 0.3% Triton X-100 in PBS) and the slides were incubated for 2 days in a cold room.Alexa Fluor 488-conjugated secondary antibody was diluted 1:1000 in blocking buffer and the slides were incubated for 2 h at room temperature to visualize both immunoreactive signals.The slides were cover-slipped and subjected to microscopy, using a BX43F Olympus fluorescent microscope (Center Valley, PA) with a DP80 camera.Preliminary experiments to determine the appropriate working concentration and incubation time for each antibody were performed.For negative controls, the primary antibodies were removed, and the slides were only incubated with the blocking buffer followed by Alexa Fluor 488-conjugated secondary antibody for the appropriate time period.

Cyclic AMP assay
Cyclic AMP (cAMP) was assayed using a direct immunoassay kit (Arbor Assays, Ann Arbor, MI), as previously described 69,71 .Briefly, hRPTCs and mRPTCs were grown in 12-well plates.The RPTCs at ~ 75% confluence were pretreated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 M; Sigma-Aldrich, St. Louis, MO, USA), before the addition of a D1-like receptor agonist, fenoldopam (1 µM/30 min), NPFF, RFRP-2, RFRP-3, and/or AC-263093, at the indicated concentrations and time, and re-challenged with forskolin (10 µM) or PBS for 30 min.The cell lysates were prepared to determine the protein concentration, using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).After quantification, the same amount of cell lysates (Supplementary Fig. S3) or culture supernatants (Figs. 3 and 6) were used to determine cAMP concentrations with reading the optical density at 450 nm by an ELISA plate reader.

Co-immunoprecipitation assay
Co-immunoprecipitation was performed using a Dynabeads kit (Thermo Fisher Scientific), as previously described 74 .Briefly, ~ 90% confluent hRPTCs were harvested, and the cell pellets were lysed in a lysis buffer (20 mM Tris•HCl, pH 8.0/1 mM EDTA/1 mM NaN3 /2 mM DTT/0.25 M sucrose), with 0.2 mM phenylmethylsulfonyl fluoride, and protease and phosphatase inhibitor cocktail.Five µg of anti-D 1 R, anti-D 5 R, anti-NPFF-R1, or anti-NPFF-R2 antibodies were conjugated with Dynabeads in 0.5 mL of slurry.The cell lysates were then incubated with the conjugated antibodies at 4 °C for 4 h, followed by proper washing.The controls were normal rabbit IgG and chicken IgY.Proteins bound to the beads were eluted in 60 µL of loading buffer at 65 °C for 15 min, separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane for incubation with the detecting antibody, followed by the appropriate secondary antibody, before visualization with a LiCor Odyssey Imaging system.

Targeted quantification of NPFF with Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Targeted LC-MS/MS method was used to measure NPFF in the mouse serum and kidney as previously described 75,76 .Briefly, C57BL/6 mouse serum and kidney samples were homogenized and sonicated in ice-cold acidified 90% methanol buffer to precipitate large proteins and extract NPFF in the supernatant.Supernatant samples were collected after centrifugation at 18,000×g for 30 min at 4 °C.Molecular weight cutoff (MWCO, 10 K) ultra-centrifugal filter (Sigma) was used to enrich molecules lower than 10 K MW, which were then dried down and desalted by Waters HLB solid phase extraction plate. 13C 5 , 15 N folic acid was spiked into the sample as the internal standard (I.S.).LC-MS-MS analysis was conducted using a Dionex Ultimate 3000 RSLCnano system coupled with a Thermo Scientific Q-Exactive HFX mass spectrometer as described previously 75 .An Easy-spray PepMap C18 LC column (2 μM, 100 Å, 75 μM × 15 cm) was used to separate peptide samples with a 1 h LC gradient.Serial concentrations of NPFF standards (Cayman) were spiked into a highly diluted sample matrix (no detectable NPFF signal) with I.S. to generate calibration curves.A parallel reaction monitoring (PRM) method was established by using the a2, b2, y4, y6 fragment ions from NPFF peptide, normalized to the fragment ion from the I.S.Data analysis was conducted with Skyline software 76 and R studio.

Urinary sodium excretion and blood pressure measurement
Adult C57BL/6 mice (male, 8-week-old), purchased from Jackson Laboratory (Bar Harbor, ME), were housed in a temperature-controlled facility with a 12:12-h light-dark cycle and fed with regular mouse chow and water ad libitum for at least 2 weeks before any studies were performed.Renal Npff-r1 and Npff-r2 were silenced by the chronic renal subcapsular infusion of specific Npff-r1 and Npff-r2 siRNA (Cat.No. SI01037379 and SI04925039 respectively, Qiagen, Germantown, MD), via an osmotic minipump, as previously described 69,71,72 .Briefly, the mice were uninephrectomized 1 week prior to the implantation of the minipump.For the minipump

Figure 2 .
Figure 2. Analysis of NPFF peptide by targeted LC-MS/MS.(a) Extracted LC-MS ion chromatograms (m/z 541.3010) of NPFF peptide standard and NPFF extracted from the mouse kidney and serum.(b) MS spectrum of NPFF peptide.(c) MS/MS peptide fragmentation spectrum of NPFF peptide detected in mouse serum (red) versus standard (blue).(d) NPFF calibration curves using a2, b2, y4, and y6 fragment ions based on the targeted parallel reaction monitoring (PRM) assay.

Figure 5 .
Figure 5. Co-immunoprecipitation of NPFF receptors with D1-like receptors (D 1 R and D 5 R) in hRPTCs.(a) Co-immunoprecipitation of NPFF-R1 and NPFF-R2 with D 1 R in hRPTCs.(b) Co-immunoprecipitation of NPFF-R1 but not NPFF-R2 with D 5 R in hRPTCs.hRPTC lysates were immunoprecipitated (IP) with anti-NPFF-R1 or anti-NPFF-R2 antibodies coupled to Dynabeads for 4 h at 4 °C.The protein complexes bound to the beads were eluted and separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted (IB) with anti-D 1 R (a) or anti-D 5 R (b) antibodies, as indicated.The expected bands for D 1 R and D 5 R are at 70 kDa and 55 kDa, respectively.Normal IgG was used for negative control and immunoblotting of D 1 R or D 5 R in cell lysates for positive control.